title: Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage.
abstract: Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix. The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7-11 d to complete.
authors.affiliation: University of Melbourne Department of Paediatrics, Parkville, Victoria, Australia.
authors.forename: Heather
authors.initials: H
authors.lastname: Stanton
authors.forename: Suzanne B
authors.initials: SB
authors.lastname: Golub
authors.forename: Fraser M
authors.initials: FM
authors.lastname: Rogerson
authors.forename: Karena
authors.initials: K
authors.lastname: Last
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Little
authors.forename: Amanda J
authors.initials: AJ
authors.lastname: Fosang
mesh_terms: D051722:ADAM Proteins / Q000378:metabolism*; D053674:Aggrecans / Q000032:analysis* / Q000378:metabolism; D000818:Animals; D001168:Arthritis / Q000473:pathology; D015153:Blotting, Western; D002356:Cartilage / Q000737:chemistry / Q000201:enzymology*; D008751:Methylene Blue / Q000031:analogs & derivatives; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic
chemical_list: D053674:Aggrecans; C435946:dimethylmethylene blue; D051722:ADAM Proteins; D008751:Methylene Blue